CS260068B1 - The strain of Pseudomonas sp. CCM 3987 high 7 / 3- (4-carboxybutanamido) cafalosporanic acid hydrolase - Google Patents

Abstract

The invention relates to a new strain of the microorganism Pseudomonas sp. CCM 3 987, producing an increased amount of an intracellular enzyme, Ίβ-(4-carboxybutanamido)cephalosporanic acid hydrolase, for the preparation of 7-aminocephem compounds, in particular 7-aminocephalosporanic acid, which is a starting intermediate for the production of semi-synthetic cephalosporins, for example cefazolin.

Description

The invention relates to a microorganism strain of Pseudomonas sp. CCM 3 987 with high production of 7-beta- (4-carboxybutanamido) cephalosporanoic acid hydrolase useful for the enzymatic preparation of 7-aminocephemic compounds, in particular for the preparation of 7-aminocephalosporanoic acid (hereinafter 7-ACK). 7-ACK is an important intermediate for the preparation of semisynthetic cephalosporin antibiotics such as cefazoline.

It is known that by the action of a specific amide bond hydrolyzing enzyme, 7-beta- (4-carboxybutanamido) cephalosporanoic acid hydrolase (hereinafter glutaryl-7-ACK), 7-aminocephene compounds of formula I ^H 2N-I-ΓΊ can be obtained

-CHjX

COOH (I) in which X represents a hydrogen atom, -OH, -OCOCH 3 or a nucleophilic group, from their 7-acylamino derivatives of formula II

A, —CO — ΝΗΟ '

COOH (II) in which R denotes - _{(CH2> 3} -COOH or - (CH _{2) 3} -CO-COOH, and X have the same meaning as in formula I.

Microbial strains with said enzyme activity can be used for the two-step enzyme technology of preparing 7-ACK from cephalosporin C, a compound of formula II wherein X is -OCOCH-j and R is - (CH3) j-CHNHj-COOH. This technology involves the enzymatic preparation of 7-acylaminocefemsloučenin of formula III wherein R denotes - _{(CH2) 3-COOH,} i.e., 7-beta- (4-carboxybutanamide) cephalosporanic acid or 3-acetometoxymetyl-7-beta- (4- carboxybutanamido) -ceph-3-em-4-carboxylic acid, and its subsequent hydrolysis.

Said 7-acylaminocephem compound can be obtained from cephalosporin C, and optionally from a cephalosporin. from its derivatives either chemically (Japanese Patent Nos. 7,777,078 and 78,112,891), or enzymatically by the action of permeabilized, resp. immobilized cells, D-amino acid oxidase producers (British Patent No. 1,272,769, Japanese Patent Nos. 7,688,694, 7,744,695, 77,125,696, 77,127,295 and 79,154,592 and Czechoslovakian author's certificate no. 251 457, depending on Czechoslovakian author's certificate no. 231 458)

Pseudomonas ovalis, Comamonas (Japanese Patent Nos. 75 101 584 and 7 670 884), Bacillus, Arthrobacter and Alcaligenes (Japanese Patent Nos. 77 128 293 and 7 886 094) are described for the hydrolysis of said 7-acylaminocephem compounds. ).

In MS. No. 239 293 describes a strain of Pseudomonas sp. CCM 3,635 exhibiting a constitutive synthesis of glutaryl-7-ACK intracellular hydrolase, so there is no need to add a glutaric acid inducer to the culture broths. Another advantage of this strain is that it does not exhibit undesirable beta-lactamase activity.

The present invention relates to a novel microbial high-production strain which has been derived from the parental strain Pseudomonas sp. CCM 3,635 by chemical mutagenesis and selection of monocolonium isolates, the advantage of which compared to the parent strain is more than twice the specific activity of glutaryl-7-ACK hydrolase, while retaining the original characteristics of the parent strain which are industrially advantageous, ie the synthesis of this enzyme in absence of inducer in the culture medium (constitutiveness) and inability to synthesize beta-lactamases. Other biochemical characteristics of the new strain do not differ from the parent strain.

The new strain is stored in MS. Czechoslovak Collection of Microorganism, J. E. Purkyně University, Brno, tř. Defenders of Peace 10, under the name Pseudomonas sp.

CCM 3,987.

. The amount of this specific hydrolase in the cells was quantitatively determined by measuring the initial rate of hydrolysis of glutaryl-7-ACK in 0.1 M phosphate buffer at pH 7.0 and 37 ° C in the region of zero-order enzyme kinetics. Reaction products were determined spectrophotometrically using p-dimethylaminobenzaldehyde (Balasingham et al., Biochim. Biophys. Acta 276, 250, 1972). One unit (U) of glutaryl-7-ACK hydrolase enzyme activity is that amount of enzyme which corresponds to the formation of one micromole (1 µm) of 7-ACK per minute under the above reaction conditions. Specific activity is defined as U. g ^-1 dry cell mass. Chromatography was used, and a thin layer of cellulose using n-propanol-water (7: 3) with ninhydrin detection (R _f 0.5) on a semiquantitative evaluation densitometer Vitatron TLD 100. The active agent content of the product was verified by high pressure liquid chromatography (HPLC ).

The subject strain Pseudomonas sp. CCM 3 987 is useful for enzymatic hydrolysis of glutaryl7-ACK prepared either chemically (by chemical synthesis) or enzymatically (by oxidation of cephalosporin C with D-amino acid oxidase). Preferably, the present high-production strain can be used as a starting source for the production of immobilized biocatalysts both on the basis of immobilized cells and for the isolation and subsequent immobilization of this specific hydrolase. The subject strain can also be used for further gene manipulation to multiply the structural genes bearing information for the synthesis of this enzyme.

The advantage of using a new strain of Pseudomonas sp. CCM 3 987 to a significantly higher specific activity of glutaryl-7-ACK hydrolase by 150% on average compared to the parent strain. The biosynthesis of this enzyme in the novel novel high-production strain ranges from 220 to 270 U at the end of the culture. liter of culture liquid and the specific activity of the cells is 50-75 U. g ^-1 dry cell mass.

These advantageous properties of the novel strain allow a reduction in time during the hydrolysis of oxidized forms of cephalosporin C, especially glutaryl-7-ACK, thereby reducing the total amount of undesirable by-products.

Furthermore, a qualitative and quantitative comparison of the microorganism strain properties with glutaryl-7-ACK hydrolase activity is presented in the genealogical scheme.

Characteristics Natural isolate of Pseudomonas sp.

Presence of beta-lactamases +

Induction of glutaric acid enzyme enzyme +

Specific activity

Pseudomonas sp. CCM 3,635

Pseudomonas sp. CCM 3,987

25_

208.3 to 75

417 to 625

In the following examples, a method for obtaining a novel high-production microorganism strain is described in detail.

Example

Pseudomonas sp. CCM 3 987 was obtained by this procedure. The culture of Pseudomonas sp. CCM 3,635 grown on sloping masopepton agar was seeded with 100 ml of liquid broth in 500 ml beakers of the following composition: 1% peptone, 0.5% beef extract, 0.1% yeast extract, 0.5% sodium chloride. The flasks were incubated on a rotary shaker at 240 rpm. min ¹ at 28 ° C for 24 hours.

ml of this culture of the first vegetative inoculum generation was used to inoculate the broth of the same composition with the same flasks and cultured under otherwise identical conditions. The second vegetative generation culture was aseptically centrifuged in the second third of the exponential phase of cell growth and the supernatant discarded. The separated cells (sediment) were washed under aseptic conditions with sterile 0.1 M phosphate buffer pH 7.0 and suspended in an equal volume of the same buffer. The prepared cell suspension was then added to a solution of N-methyl-N'-nitro-N-nitro7. 8 - i soguanidine in the same buffer so that the resulting mixture contains about 10 to 10 cells per ml and 150 µg of said mutagen.

The mixture was stirred at room temperature for 60 minutes and then plated on petri dishes containing solid broth with masopepton agar. After a three-day incubation at 28 ° C, grown colonies were transferred to solid soil on a solid soil on petri dishes containing 0.5% protein hydrolyzate (commercial product Vitana), 0.5% yeast extract, 0.1% corn extract and 0.3% glutaric acid p-nitroanilide (sterilized by filtration through a sterile Seitz filter) and 2% agar.

After a 14-day incubation at 28 ° C, a selection of monocolon isolates (clones) was selected, the principle being that the smallest colonies were inoculated on mosopepton slant agar.

Thereafter, the individual isolates, after their submersible cultivation in flasks as described above and in a liquid broth containing 1% peptone and 25% filtrate of a 4% corn extract solution (which had been pre-precipitated at boiling at pH 7.6), were again described above. mutagenized by the same chemical mutagen.

The mutagenesis suspension was plated on solid non-selective masopepton broth in Petri dishes and passively selected for monocolon isolates that were tested by submersive single-flask culture as follows.

A 500 ml beaker was filled with 100 ml of a liquid broth of the above composition (containing peptone and pre-precipitated corn extract) into which monocolon isolates from sloping masopepton agar were seeded by double mutagenesis and targeted and passive selection. The flasks were incubated on a rotary shaker in the manner and under the conditions previously indicated for 24 hours.

After that time, 5 ml of the culture of the first vegetative generation thus prepared was used to inoculate 100 ml of liquid culture medium in 500 ml boiling flasks consisting of: 5% protein hydrolyzate (commercial product Vitana) and 50 ml filtrate of a 4% corn extract solution precipitated pre boiling at pH 7.6. The second vegetative generation cultures were cultured under otherwise identical conditions for 42 hours. Then the cultures were separated by centrifugation (16,000 G for 10 minutes), the supernatant discarded, the cell sediment washed with an equal volume of 0.1 M phosphate buffer pH 7.0, and standard cell suspensions in the same buffer containing 8 mg dry cell mass were prepared. ml ^-7 . These suspensions were further evaluated for the specific enzyme activity of glutaryl-7-ACK hydrolase as compared to a control suspension of parental strain cells which also grew under the same culture conditions, the reaction conditions of the assay being described in the description of the present invention.

From a series of monoclonal isolates (clones) that were subjected to quantitative enzyme analysis as described, only one of them achieved specific activity in the range of 50-75 U. g ^-1 , which is 100 to 200% higher specific activity compared to the parental strain.

Claims (1)

Pseudomonas species strain CCM 3 987 with high production of 7-beta- (4-carboxybutanamido) cephalosporanoic acid hydrolase.